Thyroid function is regulated by TSH secreted by the pituitary (Szkudlinski M W, et al 2002. Physiological Reviews 82: 473-502). TSH binds to the TSHR on the surface of thyrocytes and this is the first step in initiating the TSHR signalling cascade. Binding of TSH to the TSHR leads to stimulation of formation and release of thyroid hormones; thyroxine (T4) and tri-iodothyronine (T3). A feedback mechanism involving the levels of T4 and T3 in the circulation and thyrotropin releasing hormone (TRH) secreted by the hypothalamus controls the release of TSH that in turn controls thyroid stimulation and the levels of thyroid hormones in serum (Szkudlinski M W, et al, 2002 supra). The TSHR is a G-protein coupled receptor and is composed of three domains:—a leucine rich repeat domain (LRD), a cleavage domain (CD) and a transmembrane domain (TMD) (Núñez Miguel R, et al 2004. Thyroid 14: 991-1011).
It is well documented in the art that some patients with autoimmune thyroid disease (AITD) develop autoantibodies which are reactive with the TSHR (Rees Smith B, et al 1988. Endocrine Reviews 9: 106-121). There are two main types of TSHR autoantibodies (TRAbs); a stimulating type and a blocking type. Thyroid stimulating type autoantibodies bind to the TSHR and mimic the actions of TSH thereby stimulating the thyroid to produce high levels of T4 and T3; these autoantibodies are also described as TRAbs with stimulating activity or TSH agonist activity (Rees Smith B, et al 2007. Thyroid 17: 923-938). The feedback control mechanism of thyroid function is no longer effective in the presence of thyroid stimulating autoantibodies and patients present with the clinical symptoms of a hyperactive thyroid characterised by an excess of thyroid hormones in serum and its metabolic consequences. This condition is known as Graves' disease. TRAbs with stimulating activity may also interact with the TSHRs in retroorbital tissue and contribute to the development of the eye signs of Graves' disease. A human monoclonal autoantibody which acts as a powerful thyroid stimulator (hMAb TSHR1; also referred to as M22) has been described in detail in WO 2004/050708A2. The structure of the complex of M22 Fab bound to the TSHR LRD has been solved by x-ray crystallography at 2.55 Å resolution as described in WO2008/025991A1. Analysis of the structure of the TSHR-M22 complex provides detailed information about the receptor residues and the stimulating autoantibody residues involved in interactions with each other.
M22 has been used in ELISA for TSHR antibody measurement (Zöphel, K et al, Clinica Chimica Acta 2009 and Zöphel, K et al, Clinica Chimica Acta 2008.
Blocking type TRAbs occur less frequently in patients with AITD than stimulating autoantibodies. Blocking type autoantibodies bind to the TSHR, prevent TSH from binding to the receptor but have no ability to stimulate TSHR activity. Consequently formation and secretion of thyroid hormones (T4 and T3) is greatly reduced and the patients with this type of TRAb can present with clinical symptoms of an under-active thyroid (hypothyroidism). Blocking type autoantibodies are known as TRAbs with blocking activity or TSH antagonist activity (Rees Smith B, et al 1988 supra and Rees Smith B, 2007 et al supra). TRAbs with blocking activity when present in serum of pregnant women cross the placenta and may block the TSHRs in the foetal thyroid leading to neonatal hypothyroidism and serious consequences for development. Furthermore, TRAbs with blocking activity can be found in breast milk of affected mothers and may cause clinical hypothyroidism in the baby (Evans C, et al 2004 European Journal of Endocrinology 150: 265-268). A human autoantibody to the TSHR with TSH antagonist activity (5C9) has been described in detail in WO 2008/099185A1. Clinical symptoms in patients with AITD and circulating TRAbs are related to the effect of autoantibodies on TSHR activity i.e. whether the TRAbs cause stimulation or blocking. It has been proposed, however, that in some patients a mixture of stimulating and blocking TRAbs may be present simultaneously with the overall clinical presentation related to higher concentration and/or activity of one type of the TRAbs (Rees Smith B et al 1988 supra; Furmaniak J et al 1993 Springer Seminars in Immunopathology 14: 309-321 and Schott M et al 2005 Trends in Endocrinology and Metabolism 16: 243-248). Furthermore, the concentrations and/or activities of stimulating or blocking TRAb may vary in the same patient during the course of the disease and indeed fluctuation of symptoms from hypo- to hyperthyroidism in the same patient over time has been reported (Rees Smith B et al 1988 supra; Furmaniak J and Rees Smith B 1993 supra and Schott M et al 2005 supra). However, attempts to separate the TRAbs with different bioactivity or to differentiate between these TRAbs in serum samples using currently available bioassays is difficult. More recently, the invention described in WO2006/016121A1 provides a means to discriminate between stimulating and blocking types of TRAbs using bioassays that employ TSHR mutated at R255.
Human recombinant TSH (Thyrogen®) is a preparation of human TSH produced under cGMP regulations as a recombinant protein and approved by the US FDA as an aid in the diagnosis of residual or recurrent thyroid cancer (Duntas L H, Cooper D S 2008 Thyroid 18: 509-516). Monitoring of thyroid cancer patients after treatment includes stimulation of thyroid remnants or metastases with recombinant human TSH followed by a thyroid scan and/or measurement of serum thyroglobulin levels (Duntas L H and Cooper D S 2008 supra). Human chorionic gonadotropin is a hormone produced during pregnancy which has mild thyroid stimulating effects (Grossmann M et al 1997 Endocrine Reviews 18: 476-501). Characterisation of stimulating or blocking types of TRAbs and how they interact with the TSHR is of critical importance for development of improved methods to diagnose and manage different forms of AITD. In addition these studies are critical for developing new strategies for the management of diseases associated with an autoimmune response to the TSHR. The availability of potent thyroid stimulators other than recombinant human TSH provides new alternatives for monitoring and managing thyroid cancer patients.